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| United States Patent |
5,578,449
|
|
Fr asch
, et al.
|
November 26, 1996
|
Procedure for the sex determination of embryos in mammals especially
applied to bovine embryos
Abstract
A procedure is provided for the sex determination of embryos in mammals,
that is particularly exemplified on bovines. The procedure overcomes the
limitations given in former techniques. It includes a new stage of
polymerase chain reaction PCR method, using specific oligonucleotides in
order to amplify fragments corresponding to the sampled cells and not from
false ones, spuriously produced. In this way, the necessary sensitivity is
obtained. The oligonucleotides sequences are particularly selected for the
procedure. The procedure is used for the sex determination of embryos in
mammals, and the procedure is sensitive enough to be carried out a sample
containing few cells, is highly secure, and which give positive results
for both female and male sex; specially applied to bovine embryos.
| Inventors:
|
Fr asch; Alberto C. (Buenos Aires, AR);
Ugalde; Rodolfo A. (Buenos Aires, AR)
|
| Assignee:
|
Hilding Ohlsson, S.A. (Buenos Aires, AR)
|
| Appl. No.:
|
425711 |
| Filed:
|
April 20, 1995 |
Foreign Application Priority Data
| Current U.S. Class: |
435/6; 435/91.2; 536/24.3; 536/24.33 |
| Intern'l Class: |
C12Q 001/68; C12P 019/34; C07H 021/04 |
| Field of Search: |
436/6
435/91.2,78
|
References Cited
U.S. Patent Documents
| 5055393 | Oct., 1991 | Kwoh et al. | 435/6.
|
| 5461145 | Oct., 1995 | Kudo et al. | 536/24.
|
Other References
A. H. Handyside et al Nature vol 344, 768 (1990) "Pregnancies from biopsied
human preimplantation embryos by specific DNA amplification".
M. W. Bradbury et al, Proc Natl Acad. Sci. 87, 4053 (1990) "Enzymatic
amplification of a Y chromosome repeat in a single blastomere allows
identification of the sex of preimplantation mouse embryos".
H. A. Ehrich et al., Science 252, 1643 (1991) "Recent Advances in the
Polymerase Chain Reaction".
J. D. Watson et al. Recombinant DNA Second Edition, Chapter 6 "The
Polymerase Chain Reaction" Scientific American Books, W. H. Freeman & Co.
(1992).
Palmer et al. Comparison Of Human ZFY And ZFX Transcripts, PNAS
87:1681-1685.
Pollevick et al. Sex Determination Of Bovine Embryos By Reastriction
Fragment Polymorphisms Of PCR Amplified ZFX/ZFY Loci, Biotechnology 10:
805-807.
Peura et al. Theriogenology 35: 547-555.
Albeut et al., J. Clin. Microbiol 28: 1560-1564.
Aurelius et al, The Lancet, 337(8735), Jan. 26, 1991, pp. 189-192.
|
Primary Examiner: Zitomer; Stephanie W.
Assistant Examiner: Rees; Dianne
Attorney, Agent or Firm: Walker; Alfred M.
Parent Case Text
This application is a continuation of application Ser. No. 07/868,995,
filed Apr. 15, 1992.
Claims
We claim:
1. A process for sex determination of embryos in bovines which comprises:
a) isolating a bovine embryo at the blastula stage;
b) cutting a zone of cells away from the pellucidae membrane of said embryo
by microsurgery and extracting a sample of about 8 to about 19 cells from
said zone of cells;
c) separating said about 8 to about 19 cells from remaining traces of the
embryo's pellucidae membrane;
d) isolating DNA from said about 8 to about 19 cells by placing the sample
in a tube with water, heating the contents of the tube to 95.degree. C. to
form an aqueous denatured DNA solution, immediately chilling the contents
of said tube by placing the tube in ice, and centrifuging the contents of
said tube to recover any condensed liquid containing the aqueous denatured
DNA solution;
e) performing a first round of a polymerage chain reaction (PCR) on said
aqueous denatured DNA solution to amplify a first fragment of DNA wherein
said first fragment of DNA is a subsequence of a sequence in a homologous
genome region of the X and Y chromosome, using a first pair of
oligonucleotide primers with the sequence 5'-ATAATCACATGGAGAGCCACAAGCT-3'
(SEQ. ID. NO:3) and 5'-CGACTTCTTTGGTATCTGAGAAAGT-3' (SEQ. ID. NO:4), in
the presence of a DNA polymerage enzyme and nucleotides until a solution
of a first amplified DNA fragment is obtained;
f) obtaining an aliquot of said solution and subjecting said aliquot to a
second round of PCR to amplify a second DNA fragment whose sequence is a
subsequence of said first amplified DNA fragment using a second pair of
oligonucleotide primers with the sequence: 5'-TTGAATGTGATGAGTGTGGG-3'
(SEQ. ID. NO: 5) and 5'-AAGTCAGAAGACAAATGTCA-3' (SEQ. ID. NO: 6) until a
solution of a second amplified DNA fragment is obtained;
g) digesting said second amplified DNA fragment with PstI, wherein the
presence of a PstI cut site in said second amplified DNA fragment
indicates the presence of a Y chromosome and wherein the absence of a PstI
cut site in said second amplified DNA fragment indicates the presence of
an X chromosome, thereby forming a mixture of cut and/or uncut second
amplified DNA fragments;
h) size separating said mixture of cut and/or uncut second amplified DNA
fragments by electrophoresis on a 2% agarose gel and determining the size
of digested fragments by a comparison of bands obtained on said gel with a
standard molecular weight marker wherein the presence of a 378 base pair
band is indicative of the presence of an X chromosome and the presence of
two bands which are 293 base pairs and 85 base pairs each is indicative of
the presence of a Y chromosome;
i) determining the sex of the embryo based on results obtained in step (h).
2. The process as in claim 1, wherein at step (g), 5 units of PstI is used
and digestion is performed for 2 hours.
Description
INTRODUCTION
1. Field of Application
The present invention refers to a novel procedure for the sex determination
of embryos in mammals, especially as applied to bovine embryos.
The improvement of cattle quality through crossbreeding of individuals with
selected hereditary characteristics is a technique that has been used for
quite a long time.
Artificial insemination techniques have lowered the costs involved since
from a sole selected male a great number of offspring can be obtained.
However, these techniques are being supplanted quickly by embryo
implantation in females, in which females are previously prepared for the
implantation of an embryo. Implantation of frozen embryos has many
advantages such as, for example, a greater breeder's performance,
especially with females, changing the pregnancy periods according to the
climatic seasons and according to commercial circumstances, as well as to
providing greater facility in national and international home trade.
Nevertheless, these techniques run into a limitation in that the embryo's
sex is not known even though it is determined at the moment of
fertilization, so the embryos must be implanted, obtaining in this way an
unpredictable result.
In the case of animals like bovines, determinating the embryo's sex will
allow one to obtain advantages in cattle commercialization and
exploitation.
2. Discussion of the Prior Art
The development of procedures to determine an embryo's sex have already
been tried, but only poor and limited results have been obtained.
Some of these procedures are based on antigen-antibody reactions.
Antibodies obtained against specific proteins which are only produced by
embryos of one sex, and not of the other sex, are allowed to react with
whole embryos and their reactivity is detected by a second antibody.
In such procedures, beside the sensitivity problems, there are
uncertainties when the result is negative. This means that, when the
result is positive, the antigen-antibody reaction guarantees that the
cells of the sample belong to a determinated sex. But when the result is
negative, it could be due to cells belonging to an embryo of the opposite
sex, or caused by mistakes or imperfections while applying the technique.
It is also possible to obtain a negative result if one of the reactants
utilized is spoiled or destroyed, because of an excess in the temperature
of the protein, an insufficient sample, etc.
The procedure of the present invention overcomes such obstacles as it gives
a definitive result for each sex, and a different result when a mistake
occurs during the application of the technique.
The rest of the experimental procedures are based, as in the present
invention, on the analysis of the DNA of cells that have been sampled. The
limitation, in the typical prior art DNA analysis procedures, comes from
the fact that the sensitivity of the prior art method isn't enough, and
the results are affected by the "noise" of molecules produced in secondary
reactions.
For a better consideration of the problem, it is enough to say that the
cells sampled never exceed 20, being normally 8 to 12 cells. A greater
number of cells removed as samples would damage the embryo from which the
cells are obtained.
Any cell of a higher mammal has about 2 pg (2.times.10.sup.-12 g of DNA.
The DNA could have about 2-3.times.10.sup.9 base pairs. But a gene (for
example, such as a sex determination gene) could have around 1000 pairs of
bases, which means that it is necessary to analyze the millionth part of
the DNA per cell.
Supposing 10 cells are sampled, the amount to be analyzed is of:
2 pg.times.10.times.10.sup.-6 =20.times.10-18 g of DNA
In a human and a mouse it is possible to use the repetitive specific
nucleotides sequences of Y chromosome, which here indicates that it is a
male embryo ([1]A. H. Handyside et al., Nature 344, 768, 1990, and [2]M.
W. Bradbury et al., Proc. Natl. Acad. Sci. 87, 4053, 1990), but these
methods have the same uncertainty as regards to the one method indicated
for antigen-antibody methods already mentioned.
Actually, the most promising procedures are based on the application of the
Polymerase Chain Reaction (PCR) technique on the DNA of the sampled cells,
since it was established that, in a homologous genome region of the X and
Y chromosomes, there are differences in their sequence, making it possible
to identify the embryo's sex ([2]). This technique, a DNA chain extension
with Polymerase ([3]) H. A. Erlich et al., Science 252, 1643, 1991),
consists of consecutive steps of amplification of a DNA fragment, in this
particular case, a fragment located in the sex chromosome X/Y. For this,
specific oligonucleotide primers are required. These techniques, described
in detail farther on, have a limitation. Although the number of DNA
fragments that will be finally detected, are doubled in each step, there
comes a moment in which, for different reasons, a number of fragments, not
related to the target fragment, is produced at the same time, and the
unrelated fragments start to mask the desired result the present invention
is looking for, i.e., such as by noise. In the case where the number of
amplification steps is increased, the background would be greater.
Taking into account the limitation stated above, such procedures are not
sensitive enough to determine the embryo's sex.
The present invention proposes a technique that overcomes the
aforementioned limitations, as it adds a second step of PCR amplification
with other oligonucleotide primers, allowing a subsequent amplification of
the material already amplified in the first step. In this way, the
necessary sensitivity is attained.
BRIEF DESCRIPTION OF THE PREFERRED EMBODIMENT
The present invention suggests a procedure with the following stages:
a) obtaining a mammal's embryo in its blastula stage of development,
cutting, by means of microsurgery, the pellucidae area and extracting a
sample containing from 8 to 19 cells, preferably from 8 to 12 cells;
b) separating by dissection the cells from the traces of the embryo's
pellucidae membrane;
c) isolating and denaturing the DNA of the cells, inserting said cells in a
tube with water, heating to 95.degree. C., chilling immediately in ice and
centrifuging to recover the condensed liquid, aqueous DNA solution;
d) submitting such aqueous DNA solution to a first round of the polymerase
chain reaction (PCR) technique with a first pair of oligonucleotides able
to act as "primers" to amplify a first fragment of DNA belonging to the
sex chromosome in the presence of a polymerase as a specific enzyme and
nucleotides, until a first amplified DNA solution is obtained;
e) submitting an aliquot of the first amplified DNA solution to a second
round of PCR with a second pair of oligonucleotides able to act as
"primers" to amplify a second fragment, internal to the first fragment
amplified in the previous stage, until a second amplified DNA solution is
obtained;
f) adding to the second amplified DNA solution a restriction enzyme, able
to cut the amplified second fragment of the sexual chromosome DNA when it
is a Y chromosome, and unable to cut it when it is an X chromosome;
g) fractionating according to size by means of electrophoresis in an
agarose gel, staining with ethidium bromide and visualizing with UV
(ultraviolet) light;
h) comparing with a standard molecular weight marker;
i) determining the embryo's sex according to the presence of only one band,
with the same number of base pairs as the the second fragment of the sex
chromosome DNA (female sex), or three bands, the first with the same
number of base pairs as the second fragment of the sex chromosome DNA, the
second and the third bands corresponding, each one, to a number of base
pairs, which, added up, will give the same number of base pairs as the
second fragment of the sex chromosome DNA (male sex);
The aforementioned first pair of oligonucleotide primers in stage d),
second pair of oligonucleotide primers in stage e) and the restriction
enzyme in stage f) are chosen beforehand in the following way:
I) determining the DNA sequence of the first fragment corresponding to the
sex X and Y chromosome in stage d), and selecting the second DNA fragment,
internal to the first fragment (without sharing any of the termini of the
first fragment);
II) choosing a restriction enzyme able to cut the Y chromosome DNA second
fragment and not cut the X chromosome DNA second fragment, according to
the base sequence determined in step I);
III) choosing a first sequence of about 20 to 30 bases starting from the
extreme 5' end of the first fragment, and a second sequence of about 20 to
30 bases starting from the extreme 3' end of the first fragment, the
aforementioned first sequence and the complement of the aforementioned
second sequence conforming to the sequence of bases of the first pair of
oligonucleotide primers, according to the base sequence determined in step
I);
IV) choosing a third sequence of about 20 to 30 bases starting from the
extreme 5' end of the second fragment, and a fourth sequence of about 20
to 30 bases starting from the extreme 3' end of the second fragment, the
third sequence and the complement of the mentioned fourth sequence
conforming to the sequence of bases of the second pair of oligonucleotide
primers, according to the base sequence determined in step I);
V) synthesizing in a "gene assembler" device, the aforementioned first and
second pairs of oligonucleotide primers.
According to what has been described, referred to as prior art, the
innovation brought to the present invention comprises mostly in the
inclusion of stage e), that is, the additional round of PCR with specific
oligonucleotide primers to amplify gene fragments corresponding to the
original cells, that is, the fragments that were obtained in amplifying
stage d).
The way in which the sequences of oligonucleotides are chosen is also a
novelty.
Nevertheless, there are new details in other stages of the procedure that,
related to bovines, were developed and optimized.
In one example, this exposition allows for the establishment of the basis
for the development of techniques for use with the embryos of other
mammals.
OBJECTS OF THE PRESENT INVENTION
It is, therefore, an object of the present invention, to provide a
procedure to determine a mammalian embryo's sex which procedure is
sensitive enough to carry out with a sample of a few cells.
Another object of the present invention is to provide a procedure to
determine, in a highly reliable way, the sex of mammalian embryos, giving
positive results for both female and male offspring, and not a confusing
negative result, due to imperfections in its application.
And, finally, another object is to provide a procedure for the sex
determination of embryos in mammals, especially applicable to bovine
embryos.
BACKGROUND OF THE PRESENT INVENTION
Given that it is necessary to amplify a DNA fragment of the sex chromosome,
to obtain enough sensitivity in order to determine the embryo's sex from a
few sampled cells, it is necessary to discuss the molecular mechanisms of
DNA amplification.
Any simple DNA chain has two terminals (or extremes), one of them with
phosphate in carbon 5' of the deoxyribose molecule (extreme 5'), the other
with hydroxyl in position 3' of the sugar (extreme 3'). At the same time,
the complementary chain has the same kind of extremes in opposing
positions, that is, the extreme 3' of a chain has the other 5' in front of
it. For this reason, it is considered that the two complementary chains of
a DNA molecule have opposite polarities and are antiparallel.
It is known that the replication of a DNA chain is produced by the
combination of a nucleotide-5'-triphosphate with the extreme 3' of the
chain. Therefore, this extreme 3' is the growing point of the DNA, and
synthesis proceds in opposite directions in both complementary chains.
A template chain is simply a DNA chain. However, the template chain doesn't
provide any extreme 3' able to promote the beginning of the growth of the
new chain that is going to be synthesized. For this reason, the synthesis
of a new DNA molecule is only possible in the presence of a nucleic acid
fragment that can provide the extreme 3'. The name given to this nucleic
acid fragment is "primer."
The DNA synthesis mechanisms discussed above require the action of a series
of enzymes that catalyze the reaction. The existence of polymerases has
been proven among the enzymes that have been identified.
The application to lab techniques of what has been discussed was perfected
in the so-called PCR (Polymerase Chain Reaction) [3]. In each step of this
technique, the number of chains obtained in the former step, is doubled,
so this number has a geometric growth.
However, the application of this technique has one limitation derived from
the synthesis, of spurious DNA fragments from the primers themselves which
don't correspond to the template that one wants to copy. This can be
attributed to contamination, as well as by other causes. But the correct
copies of the template, as well as the false ones, have in their terminals
the sequences of the primers themselves, which create complementary
sequences that, in the next step, will be amplified. Therefore, in the
following steps the target chains that are under consideration, as well as
the false ones, amplify, and there comes a point where it is impossible to
improve the sensitivity.
In the present invention's procedure, the polymerase chain reaction PCR
technique is improved as follows.
After the first round of the polymerase chain reaction PCR technique on a
DNA fragment, there comes a point in which it is impossible to improve the
sensitivity of the technique. Experimentally, it was found that
approximately 45 cycles is the upper limit of amplification.
To improve the relation between the number of similar chains of the
original target and the number of false chains, it is necessary to
distinguish the first ones, as the second ones won't necessarily be
homogeneous. According to the present invention it is possible to do it
looking for a smaller DNA fragment, whose sequence is a subsequece of the
original sequence. For that purpose, other DNA chains, whose sequences
correspond to sequences of the chains under consideration, are used as new
primers. In this way, the polymerase chain reaction PCR technique is
applied again, with its geometric growth, but only on the DNA amplified
fragments that correspond to the original template.
DETAILED DESCRIPTION OF THE DRAWING
FIG. 1 shows the sequence (SEQ. ID. NO: 1 and 2) corresponding to a
fragment of sex chromosomes experimentally obtained, superimposed by
indications illustrating the choice of primers for the procedures of the
present invention.
DETAILED DESCRIPTION
A typical example started with the use of bovine embryos in the blastula
stage; and a sample of cells was obtained by microsurgery. The remaining
pellucidae zone is put aside by dissection, as this area doesn't
correspond to the embryo.
The sampled cells of the embryo were introduced in a tube with 30 .mu.l of
water and the sampled cells were denatured by heating them at 95.degree.
C., cooling immediately with ice and centrifuging to recover the condensed
liquid.
To the aqueous DNA solution obtained in this way PCR was applied, but in
two rounds with different oligonucleotide primer sequences.
To get to this point in the procedure, it was necessary to choose the
sequence and synthesize the oligonucleotide primers, and for that reason
the following steps were taken, in relation with FIG. 1.
In FIG. 1, shows the sequence of bases corresponding to a fragment of
bovine chromosome, from the homologous genome region experimentally
obtained, over which other indications have been superimposed, which
indications illustrate the way in which the restriction enzyme and the
sequence of bases belonging to the oligonucleotides used as primers have
been chosen.
The fringe of two lines (in the FIG. 1) must be taken as continuous and
read from left to right and from upwards to downwards, like reading usual
writing.
In the upper line, a sequence of bases of one of the strands of a DNA
fragment of the Y chromosome is shown, from extreme 5' end to the extreme
3' end, whereas in the lower line the same was done with the strand
corresponding to that chromosome. The complementary chains have not been
represented.
The bases have been symbolized as usual as follows:
A=adenine, T=thymine, G=guanine and C=cytosine. The symbols ? and X
represent different types of indeterminations. The complementary strands
not illustrated are corresponded base to base, such as base A from one,
with base T from the other, and base G from one with base C from the
other. Besides, they are antiparallel, that is, the extreme that
corresponds to -5'- of the illustrated chains is the -3'- of the
complementary, and visa versa.
It is possible to appreciate that the sequences of chromosome Y and
chromosome X differ in some bases, whereas in the majority of bases in the
homologous region they coincide.
The following step was to look for a restriction enzyme able to cut the Y
chromosome fragment at a certain point but unable to do so on the X
chromosome fragment. After many tests, the enzyme PstI was chosen, which
is able to distinguish the sequence CTGCAG, referred to with the letter
-.varies.-, and cut it between bases C and T. On the other hand, this
sequence has got as its complement the sequence GACGTC, but this sequence,
read in the direction 5'3' of the complement, is the same sequence CTGCAG,
which is also cut by the enzyme PstI between the C and the T. But, in the
corresponding position, the X chromosome has the sequence CCGCAG which is
not recognized by the enzyme.
The advantage of choosing the enzyme PstI is that the sequence it
recognizes doesn't repeat all over the amplified fragment, and as a
consequence there is only one cut in the fragment obtained from Y
chromosome and none in the fragment obtained from the X chromosome.
Sequences of extreme bases, referred to as -XY/1- and its complementary
-XY/2- were chosen for the amplification (the last one -XY/2- is read from
right to left, as it corresponds to the antiparallel chain, and therefore
from its extreme 5' to 3'): for example, 5'-ATAATCACATGGAGAGCCACAAGCT-3'
(SEQ. ID. NO: 3) and 5'-GCACTTCTTTGGTATCTGAGAAAGT-3', (SEQ. ID. NO: 4) are
the first pair of oligonucleotide primers.
The extremes -T- of both are the 3' extremes from which the DNA fragments
are copied.
To be able to further amplify the fragments that are a copy of the X or Y
chromosome, as a result of the PCR amplification, and not copies of any
unrelated DNA fragment, the same primers cannot be used again, because all
of the fragments would be copied, instead of only the desired target
fragments. Such unrelated DNA fragments may originate from contaminants.
The second round of PCR was carried out with primers that were taken from
the extremes of an internal fragment belonging to the selected fragment in
the first round of amplification and excluding sequences from the first
oligonucleotide primers. In this way, the -N/XY 1- sequence and the
complement to -N/XY 2- (and in inverse order), as shown in FIG. 1 as
follows: 5'-TTGAATGTGATGAGTGTGGG-3' and 5'-AAGTCAGAAGACAAATGT-CA-3', (SEQ.
ID. NO: 5 and 6) were chosen, obtaining the second pair of oligonucleotide
primers.
The oligonucleotide primers for the first, as well as for the second PCR
reaction were synthesized in a "gene assembler" device.
Returning to the PCR technique, to the aqueous DNA solution 20 .mu.l of
buffer was added, containing in a final concentration of 10 mM of
potassium chloride, 10mM of ammonium sulfate, 20 mM tris pH 8.8, 2 mM of
magnesium sulfate, 0.1% of triton X-100, 100 .mu.g/ml of bovine albumin,
0.2 mM of nucleotide dTTP, 0.2 mM of nucleotide dCTP, 0.2 mM of nucleotide
dATP, 0.2 mM of nucleotide dGTP, 300 .mu.g of each one of the first pair
of oligonucleotides, and 1.5 units of the DNA polymerase enzyme of Thermus
aquaticus;
covering with 50 .mu.l of mineral oil;
repeating 45 times the following cycle:
denaturing for 30 seconds at 93.degree. C.;
letting oligonucleotide primers anneal to the homologous regions of the DNA
to be amplified for 30 seconds at 60.degree. C.;
amplifying for one minute at 72.degree..
The first solution of amplified DNA is thus obtained.
Then follows the second round of the polymerase chain reaction, which
includes the next steps:
taking one aliquot of 30 .mu.l of the amplified DNA's first solution;
adding 20 .mu.l of buffer to the tube, containing, in final concentration,
10 mM of potassium chloride, 10 mM of ammonium sulfate, 20 mM Tris pH 8.8,
2 mM of magnesium sulfate, 0.1% of Triton X-100, 100 .mu.g/ml of bovine
albumin, 0.2 mM of dTTP, 0.2 mM of dCTP, 0.2 mM of dATP, 0.2 mM of dGTP,
300 .mu.g of each one of the second pair of oligonucleotides, and 1.5
units of the DNA polymerase enzyme of Thermus aquaticus;
covering with 50 .mu.l of mineral oil;
repeating 45 times the following cycle denaturing during 30 seconds at
93.degree. C.;
letting oligonucleotides anneal to the homologous regions of the DNA to be
amplified during 30 seconds at 60.degree. C.;
amplifying for one minute at 72.degree. C.;
(In this way, the second solution of amplified DNA is obtained.)
adding 5 units of the restriction enzyme Pst1, digesting for two hours;
fractionating according to size subjecting the samples to electrophoresis
on a 2% agarose gel, at 100 volts for 2 hours; staining with ethidium
bromide and visualizing with UV (ultraviolet) light, and comparing bands
obtained with a standard molecular weight marker.
If the embryo's sex would have been female, it would have 2 X chromosomes
per cell and one only band would be obtained, corresponding to 378 bases
pairs. This is shown in FIG. 1. The 378 base pairs correspond to the
fragment that begins at the outer extreme of -N/XY 1-, up to the outer
extreme of -N/XY 2- of the X chromosome. If, on the other hand, the
embryo's sex would have been male, containing therefore one X chromosome
and one Y chromosome per cell, there would have been a first band of 378
base pairs bases (corresponding to the X chromosome), a second band of 293
base pairs (between the extreme of -N/XY 1- and the cut -.varies.- made by
Pst1) and a third band of 85 base pairs (between the cut -.varies.- and
the extreme of -N / XY 2 -) (corresponding to the Y chromosome).
It is obvious that an enzyme which produces a cut in the DNA chair of the X
chromosome and not in the DNA of the Y chromosome would also allow the
determination of the sex according to the present invention, but with the
following difference:
In the case of a female embryo, there would be obtained 2 bands with a
number of base pairs which add up and which would correspond to the
internal fragment; in the case of a male embryo, there would be obtained 3
bands.
In this way, an example of the performance of the procedure to determine
the sex of mammal's embryos has been described as an embodiment of the
present invention, as depicted by the following claims.
It is to be noted, that other modifications to the present invention may be
obtained, without departing from the spirit and scope of the present
invention, as noted in the appended claims.
__________________________________________________________________________
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(iii) NUMBER OF SEQUENCES: 6
(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 447 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: genomic DNA
(iv) ANTI-SENSE: no
(vi) ORIGINAL SOURCE:
(A) ORGANISM: bovine
(B) STRAIN: European races
(C) INDIVIDUAL ISOLATE: male bovine
(D) DEVELOPMENTAL STAGE: adults
(E) HAPLOTYPE: diploide
(F) TISSUE TYPE: blood
(G) CELL TYPE: nucleated cell
(vii) IMMEDIATE SOURCE:
(A) LIBRARY: genomic
(B) CLONE: ZFY clones obtained by PCR amplification techniques
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT: chromosome Y/short arm
(B) MAP POSITION: not determined
(ix) FEATURE:
(A) NAME/KEY: sequence of bases of one of the chains of a DNA
fragment of chromosome Y
(B) LOCATION: not determined
(C) IDENTIFICATION METHOD: it was cloned and identified by
using information corresponding to homologous sequences
present in human genome
(D) OTHER INFORMATION: it contains polymorphisms that allow
sex determination
(x) PUBLICATION INFORMATION:
(A) AUTHORS: Pollevick, Guido D.
Giambiagi, Susana
Mancardi, Sabrina
De Luca, Leonardo
Burrone, Oscar
Frash, Alberto C.
Ugalde, Rodolfo A.
(B) TITLE: Sex determination of bobine embryos by restriction
fragment polymorphisms of PCR amplified ZFX/ZFY loci
(C) JOURNAL: Bio/Technology
(D) VOLUME: 10
(E) ISSUE: No. 7
(F) PAGES: 805 to 807
(G) DATE: July 1992
(K) RELEVANT RESIDUES IN SEQ ID NO: from 1 to 447
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
ATAATCACATGGAGAGCCACAAGCTTACCAGCAAGTCAGAGAAGGCCATCGAATGTGATG60
ACTGTGGGAAGCATTTCTCCCATGCTGGGGCTTTGTTCACTCACAAAATGGTGCATAAGG120
AAAAAGGAGCCAGCAAAATGCATAAATGTAAATTCTGTGAGTATGAGACAGCTGAACAAG180
GGTTATTAAATCGCCACCTTTTGGCAGTCCACAGCAAGAACTTTCCCCATATATGTGTAG240
AGTGTGGTAAAGGTTTTCGTCACCCATCAGAGCTCAAAAAGCACATGCGAATCCATACTG300
GAGAGAAACCGTACCAATGCCAGTACTGCGAATATAGGTCTGCAGACTCTTCTAATTTGA360
AGACGCATGTGAAAACTAAGCATAGTAAAGAAATGTCTTTCAAGTGTGACATTTGTCTTC420
TGACTTTCTCAGATACCAAAGAAGTGC447
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 447 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: genomic DNA
(iv) ANTI-SENSE: no
(vi) ORIGINAL SOURCE:
(A) ORGANISM: bovine
(B) STRAIN: European races
(C) INDIVIDUAL ISOLATE: male and female bovine
(D) DEVELOPMENTAL STAGE: adults
(E) HAPLOTYPE: diploide
(F) TISSUE TYPE: blood
(G) CELL TYPE: nucleated cell
(vii) IMMEDIATE SOURCE:
(A) LIBRARY: genomic
(B) CLONE: ZFX clones obtained by PCR amplification techniques
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT: chromosome X/short arm
(B) MAP POSITION: not determined
(ix) FEATURE:
(A) NAME/KEY: sequence of bases of one of the chains of a DNA
fragment of chromosome X
(B) LOCATION: not determined
(C) IDENTIFICATION METHOD: it was cloned and identified by
using information corresponding to homologous sequences
present in human genome
(D) OTHER INFORMATION: it contains polymorphisms that allow
sex determination
(x) PUBLICATION INFORMATION:
(A) AUTHORS: Pollevick, Guido D.
Giambiagi, Susana
Mancardi, Sabrina
De Luca, Leonardo
Burrone, Oscar
Frash, Alberto C.
Ugalde, Rodolfo A.
(B) TITLE: Sex determination of bobine embryos by restriction
fragment polymorphisms of PCR amplified ZFX/ZFY loci
(C) JOURNAL: Bio/Technology
(D) VOLUME: 10
(E) ISSUE: No. 7
(F) PAGES: 805 to 807
(G) DATE: July 1992
(K) RELEVANT RESIDUES IN SEQ ID NO: from 1 to 447
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
ATAATCACATGGAGAGCCACAAGCTTACCAGCAAGGCGGAGAAGGCCATTGAATGCGATG60
AGTGCGGGAAGCATTTCTCTCATGCTGGGGCTTTGTTTACTCATAAAATGGTGCATAAGG120
AAAAAGGAGCTAACAAAATGCACAAATGTAAATTCTGTGAATACGAGACAGCTGAACAAG180
GGTTACTGAATCGCCACCTTTTGGCGGTCCATAGCAAGAACTTTCTCCATATACGTGTGG240
AGTGTGGTAAAGGTTTTCGTCATCCATCAGAGCTCAAAAAGCACATGCGAATCCATACTG300
GCGAGAAGCCGTACCAGTGCCAGTACTGCGAATATAGGTCCGCAGACTCTTCTAACTTGA360
AAACGCATGTAAAAACTAAGCATAGTAAAGAGATGCCATTCAAGTGTGACATTTGTCTTC420
TGACTTTCTCAGATACCAAAGAAGTGC447
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: synthetic nucleic acid
(A) DESCRIPTION:
The sequence was choiced following the rules of the
present invention (applied for bovines) and was
synthesized in a 'gene assembler'device, to be one of
the primers for the first amplification stage.
(iv) ANTI-SENSE: no
(vi) ORIGINAL SOURCE:
(A) ORGANISM: bovine
(B) STRAIN: European races
(C) INDIVIDUAL ISOLATE: male bovine
(D) DEVELOPMENTAL STAGE: adults
(E) HAPLOTYPE: diploide
(F) TISSUE TYPE: blood
(G) CELL TYPE: nucleated cell
(vii) IMMEDIATE SOURCE:
(A) LIBRARY: genomic
(B) CLONE: ZFY clones obtained by PCR amplification techniques
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT: chromosome X or Y/short arm
(B) MAP POSITION: not determined
(C) UNITS: bases
(ix) FEATURE:
(A) NAME/KEY: one of oligonucleotides of the first pair,
complementary with the extreme 3'of one of the
complementary chains of the first fragment of the sexual
chromosome of bovines (sequences No. 1 and 2)
(D) OTHER INFORMATION: it allow amplification of the first
fragment of the sexual chromosome of bovines.
(x) PUBLICATION INFORMATION:
(A) AUTHORS: Pollevick, Guido D.
Giambiagi, Susana
Mancardi, Sabrina
De Luca, Leonardo
Burrone, Oscar
Frash, Alberto C.
Ugalde, Rodolfo A.
(B) TITLE: Sex determination of bobine embryos by restriction
fragment polymorphisms of PCR amplified ZFX/ZFY loci
(C) JOURNAL: Bio/Technology
(D) VOLUME: 10
(E) ISSUE: No. 7
(F) PAGES: 805 to 807
(G) DATE: July 1992
(K) RELEVANT RESIDUES IN SEQ ID NO: from 1 to 25
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
ATAATCACATGGAGAGCCACAAGCT25
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: synthetic nucleic acid
(A) DESCRIPTION:
The sequence was choiced following the rules of the
present invention (applied for bovines) and was
synthesized in a 'gene assembler'device, to be one of
the primers for the first amplification stage.
(iv) ANTI-SENSE: no
(vi) ORIGINAL SOURCE:
(A) ORGANISM: bovine
(B) STRAIN: European races
(C) INDIVIDUAL ISOLATE: male bovine
(D) DEVELOPMENTAL STAGE: adults
(E) HAPLOTYPE: diploide
(F) TISSUE TYPE: blood
(G) CELL TYPE: nucleated cell
(vii) IMMEDIATE SOURCE:
(A) LIBRARY: genomic
(B) CLONE: ZFY clones obtained by PCR amplification techniques
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT: chromosome X or Y/short arm
(B) MAP POSITION: not determined
(C) UNITS: bases
(ix) FEATURE:
(A) NAME/KEY: one of oligonucleotides of the first pair,
complementary with the extreme 3'of one of the
complementary chains of the first fragment of the sexual
chromosome of bovines (sequences No. 1 and 2)
(D) OTHER INFORMATION: it allow amplification of the first
fragment of the sexual chromosome of bovines.
(x) PUBLICATION INFORMATION:
(A) AUTHORS: Pollevick, Guido D.
Giambiagi, Susana
Mancardi, Sabrina
De Luca, Leonardo
Burrone, Oscar
Frash, Alberto C.
Ugalde, Rodolfo A.
(B) TITLE: Sex determination of bobine embryos by restriction
fragment polymorphisms of PCR amplified ZFX/ZFY loci
(C) JOURNAL: Bio/Technology
(D) VOLUME: 10
(E) ISSUE: No. 7
(F) PAGES: 805 to 807
(G) DATE: July 1992
(K) RELEVANT RESIDUES IN SEQ ID NO: from 1 to 25
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
GCACTTCTTTGGTATCTGAGAAAGT25
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: synthetic nucleic acid
(A) DESCRIPTION:
The sequence was choiced following the rules of the
present invention (applied for bovines) and was
synthesized in a 'gene assembler'device, to be one of
the primers for the second amplification stage.
(iv) ANTI-SENSE: no
(vi) ORIGINAL SOURCE:
(A) ORGANISM: bovine
(B) STRAIN: European races
(C) INDIVIDUAL ISOLATE: male bovine
(D) DEVELOPMENTAL STAGE: adults
(E) HAPLOTYPE: diploide
(F) TISSUE TYPE: blood
(G) CELL TYPE: nucleated cell
(vii) IMMEDIATE SOURCE:
(A) LIBRARY: genomic
(B) CLONE: ZFY clones obtained by PCR amplification techniques
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT: chromosome X or Y/short arm
(B) MAP POSITION: not determined
(C) UNITS: bases
(ix) FEATURE:
(A) NAME/KEY: one of oligonucleotides of the second pair,
complementary with the extreme 3'of one of the
complementary chains of the second fragment (internal
to the first fragment) of the sexual chromosome of
bovines.
(D) OTHER INFORMATION: it allow amplification of the second
fragment of the sexual chromosome of bovines.
(x) PUBLICATION INFORMATION:
(A) AUTHORS: Pollevick, Guido D.
Giambiagi, Susana
Mancardi, Sabrina
De Luca, Leonardo
Burrone, Oscar
Frash, Alberto C.
Ugalde, Rodolfo A.
(B) TITLE: Sex determination of bobine embryos by restriction
fragment polymorphisms of PCR amplified ZFX/ZFY loci
(C) JOURNAL: Bio/Technology
(D) VOLUME: 10
(E) ISSUE: No. 7
(F) PAGES: 805 to 807
(G) DATE: July 1992
(K) RELEVANT RESIDUES IN SEQ ID NO: from 1 to 20
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
TTGAATGTGATGAGTGTGGG20
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: synthetic nucleic acid
(A) DESCRIPTION:
The sequence was choiced following the rules of the
present invention (applied for bovines) and was
synthesized in a 'gene assembler'device, to be one of
the primers for the second amplification stage.
(iv) ANTI-SENSE: no
(vi) ORIGINAL SOURCE:
(A) ORGANISM: bovine
(B) STRAIN: European races
(C) INDIVIDUAL ISOLATE: male bovine
(D) DEVELOPMENTAL STAGE: adults
(E) HAPLOTYPE: diploide
(F) TISSUE TYPE: blood
(G) CELL TYPE: nucleated cell
(vii) IMMEDIATE SOURCE:
(A) LIBRARY: genomic
(B) CLONE: ZFY clones obtained by PCR amplification techniques
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT: chromosome X or Y/short arm
(B) MAP POSITION: not determined
(C) UNITS: bases
(ix) FEATURE:
(A) NAME/KEY: one of oligonucleotides of the second pair,
complementary with the extreme 3'of one of the
complementary chains of the second fragment (internal
to the first fragment) of the sexual chromosome of
bovines.
(D) OTHER INFORMATION: it allow amplification of the second
fragment of the sexual chromosome of bovines.
(x) PUBLICATION INFORMATION:
(A) AUTHORS: Pollevick, Guido D.
Giambiagi, Susana
Mancardi, Sabrina
De Luca, Leonardo
Burrone, Oscar
Frash, Alberto C.
Ugalde, Rodolfo A.
(B) TITLE: Sex determination of bobine embryos by restriction
fragment polymorphisms of PCR amplified ZFX/ZFY loci
(C) JOURNAL: Bio/Technology
(D) VOLUME: 10
(E) ISSUE: No. 7
(F) PAGES: 805 to 807
(G) DATE: July 1992
(K) RELEVANT RESIDUES IN SEQ ID NO: from 1 to 20
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
AAGTCAGAAGACAAATGTCA20
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